We evaluated the influence of sampling technique (cannulation vs. stomach tube) and site (dorsal sac vs. ventral sac) on the rumen microbiome and fermentation parameters in Hanwoo steers. Rumen samples were collected from three cannulated Hanwoo steers via both a stomach tube and cannulation, and 16...
We evaluated the influence of sampling technique (cannulation vs. stomach tube) and site (dorsal sac vs. ventral sac) on the rumen microbiome and fermentation parameters in Hanwoo steers. Rumen samples were collected from three cannulated Hanwoo steers via both a stomach tube and cannulation, and 16S rRNA gene amplicons were sequenced on the MiSeq platform to investigate the rumen microbiome composition among samples obtained via 1) the stomach tube, 2) dorsal sac via rumen cannulation, and 3) ventral sac via rumen cannulation. A total of 722,001 high-quality 16S rRNA gene sequences were obtained from the three groups and subjected to phylogenetic analysis. There was no significant difference in the composition of the major taxa or alpha diversity among the three groups (p>0.05). Bacteroidetes and Firmicutes represented the first and second most dominant phyla, respectively, and their abundances did not differ among the three groups (p>0.05). Beta diversity principal coordinate analysis also did not separate the rumen microbiome based on the three sample groups. Moreover, there was no effect of sampling site or method on fermentation parameters, including pH and volatile fatty acids (p>0.05). Overall, this study demonstrates that the rumen microbiome and fermentation parameters are not affected by different sampling techniques and sampling sites. Therefore, a stomach tube can be a feasible alternative method to collect representative rumen samples rather than the standard and more invasive method of rumen cannulation in Hanwoo steers.
We evaluated the influence of sampling technique (cannulation vs. stomach tube) and site (dorsal sac vs. ventral sac) on the rumen microbiome and fermentation parameters in Hanwoo steers. Rumen samples were collected from three cannulated Hanwoo steers via both a stomach tube and cannulation, and 16S rRNA gene amplicons were sequenced on the MiSeq platform to investigate the rumen microbiome composition among samples obtained via 1) the stomach tube, 2) dorsal sac via rumen cannulation, and 3) ventral sac via rumen cannulation. A total of 722,001 high-quality 16S rRNA gene sequences were obtained from the three groups and subjected to phylogenetic analysis. There was no significant difference in the composition of the major taxa or alpha diversity among the three groups (p>0.05). Bacteroidetes and Firmicutes represented the first and second most dominant phyla, respectively, and their abundances did not differ among the three groups (p>0.05). Beta diversity principal coordinate analysis also did not separate the rumen microbiome based on the three sample groups. Moreover, there was no effect of sampling site or method on fermentation parameters, including pH and volatile fatty acids (p>0.05). Overall, this study demonstrates that the rumen microbiome and fermentation parameters are not affected by different sampling techniques and sampling sites. Therefore, a stomach tube can be a feasible alternative method to collect representative rumen samples rather than the standard and more invasive method of rumen cannulation in Hanwoo steers.
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문제 정의
[7]. Furthermore, the objective of this study was to compare the composition of the rumen microbiome and the fermentation parameters of the rumen fluid in Hanwoo cattle between samples collected using the modified stomach tube and cannulation.
제안 방법
The number of OTUs was normalized by subsampling 70,000 sequences from each rumen sample, and used to build a phylogenetic tree with the FastTree program [17]. The alpha diversity was determined through various metrics (number of OTUs, Chao1, PD_whole_tree distance, and Shannon diversity index), and the beta diversity was determined through evaluation of the principal coordinate analysis (PCoA) plot on the unweighted UniFrac distance matrix.
데이터처리
1 Means among the three sample groups were compared using ANOVA followed by Tukey’s test.
The mean proportion of each taxon identified among the total sequences and the mean abundances of fermentation parameters of the rumen fluid were compared among the three sample groups using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons post-hoc test in XLSTAT statistical software (Addinsoft, USA).
이론/모형
Metagenomic DNA was extracted from the nine rumen samples using the RBB+C bead-beating method [10]. The V3-V4 region of 16S rRNA genes was amplified from each DNA sample with the universal primers 341F and 805R [11].
All non-chimeric sequences were classified into taxa using the Greengenes reference database [15], and operational taxonomic units (OTUs) were calculated at a 97% sequence similarity threshold using the uclust program [16]. The number of OTUs was normalized by subsampling 70,000 sequences from each rumen sample, and used to build a phylogenetic tree with the FastTree program [17]. The alpha diversity was determined through various metrics (number of OTUs, Chao1, PD_whole_tree distance, and Shannon diversity index), and the beta diversity was determined through evaluation of the principal coordinate analysis (PCoA) plot on the unweighted UniFrac distance matrix.
성능/효과
Although most of the major taxa identified were not affected by the different sampling techniques, the proportions of three putative taxa, S24-7, RFN20, and SHD-231, significantly differed among the three sampling approaches. Because these taxa were only putatively classified based on sequences recovered from uncultured bacteria, they may not be well-defined and should be newly reclassified to novel taxa with the update of reference taxonomy databases in the future.
Four major genera of Bacteroidetes were identified, including Prevotella (18.3–53.4%), YRC22 (0.7–2.4%), CF231 (0.8–1.4%), and BF311 (0.1–0.5%), with no difference in proportion (p> 0.05) among the three sample groups (Fig. 2).
In conclusion, this study demonstrates that use of a stomach tube and sampling from different sites (ventral sac vs. dorsal sac) do not affect the composition of the rumen microbiome or the fermentation parameters of rumen fluid collected from Hanwoo steers.
Proportions of these six major families did not differ (p> 0.05) among the three sample groups, except for putative family S24-7, which was greater (p< 0.05) in the DS group than in the ST group.
The Bacteroidetes sequences were assigned to six major families, including Prevotellaceae (18.3–53.5%), Paraprevotellaceae (1.8–4.6%), S24-7 (1.2–4.8%), RF16 (0.3–2.0%), BS11, and Bacteroidaceae (0.2–0.5%), that accounted for ≥0.2% of the total sequences across the nine samples on average (Table S1).
The Firmicutes sequences were assigned to seven major families, including Ruminococcaceae (4.0–11.3%), Lachnospiraceae (4.9–17.1%), Veillonellaceae (6.3–12.5%), Erysipelotrichaceae (1.0–1.3%), Clostridiaceae (0.3–1.5%), Christensenellaceae (0.1–2.3%), and Mogibacteriaceae (0.3–1.6%), that accounted for ≥0.2% of the total sequences on average across the nine samples, with no difference (p> 0.05) among the three sample groups.
The proportion of the putative genus SHD-231 placed within Anaerolinaceae was also greater (p< 0.05) in the DS group than in the ST group (Fig. 2).
The proportions of TM7, Proteobacteria, Actinobacteria, Spirochaetes, Tenericutes, Chloroflexi, Planctomycetes, Synergistetes, and WPS-2 accounted for 0.2–0.8% of the total sequences on average across the nine samples with no difference (p> 0.05) among the three sample groups detected, except for Chloroflexi, which was greater (p< 0.05) in the DS group than in the ST group.
The proportions of the remaining four major genera, Desulfovibrio, Treponema, Anaeroplasma, and TG5, among the 10 phyla also did not differ (p> 0.05) among the three sample groups (Fig. 2).
The remaining 10 major phyla identified included the 10 major families RFP12 (0.5–1.1%), F16 (0.6–0.9%), Desulfovibrionaceae (0.1–0.5%), Succinivibrionaceae (0.0– 0.3%), Coriobacteriaceae (0.2–1.2%), Spirochaetaceae (0.2–0.7%), Anaeroplasmataceae (0.1–0.4%), Anaerolinaceae (0.2–1.0%), Pirellulaceae (0.2–0.9%), and Desulfovibrionaceae (0.1–0.6%) (Table S1), with no difference (p> 0.05) among the three sample groups, except for Anaerolinaceae, which showed a greater proportion (p< 0.05) in the DS group than in the ST group.
The remaining eight minor phyla identified, Lentisphaerae, SR1, Armatimonadetes, Fibrobacteres, LD1, Elusimicrobia, Fusobacteria, and GN02, accounted for <0.2% of the total sequences across the nine samples on average.
There were 14 major genera of Firmicutes identified, including Succiniclasticum (2.7–7.8%), Butyrivibrio (1.1–9.1%), Ruminococcus (0.4–3.0%), and Selenomonas (0.3–3.3%), along with RFN20, Anaerostipes, Anaerovibrio, Pseudobutyrivibrio, Clostridium, Moryella, p-75-a5, Coprococcus, Oscillospira, and Blautia, that accounted for ≥0.2% of the total sequences across the nine samples on average, with no difference (p> 0.05) among the three sample groups, except for the genera RFN20 and Blautia (Fig. 2).
참고문헌 (19)
Kim M, Morrison M, Yu Z. 2011. Status of the phy logenetic diversity census of ruminal microbiomes. FEMS Microbiol. Ecol. 76: 49-63.
Terre M, Castells L, Fabregas F, Bach A. 2013. Short communication: comparison of pH, volatile fatty acids, and microbiome of rumen samples from preweaned calves obtained via cannula or stomach tube. J. Dairy Sci. 96: 5290-5294.
Ramos-Morales E, Arco-Perez A, Martin-Garcia AI, Yanez-Ruiz DR, Frutos P, Hervas G. 2014. Use of stomach tubing as an alternative to rumen cannulation to study ruminal fermentation and microbiota in sheep and goats. Anim. Feed Sci. Technol. 198: 57-66.
Paz HA, Anderson CL, Muller MJ, Kononoff PJ, Fernando SC. 2016. Rumen bacterial community composition in Holstein and Jersey cows is different under same dietary condition and is not affected by sampling method. Front. Microbiol. 7: 1206.
Shen JS, Chai Z, Song LJ, Liu JX, Wu YM. 2012. Insertion depth of oral stomach tubes may affect the fermentation parameters of ruminal fluid collected in dairy cows. J. Dairy Sci. 95: 5978-5984.
Duffield T, Plaizier JC, Fairfield A, Bagg R, Vessie G, Dick P, et al. 2004. Comparison of techniques for measurement of rumen pH in lactating dairy cows. J. Dairy Sci. 87: 59-66.
Herlemann DP, Labrenz M, Jurgens K, Bertilsson S, Waniek JJ, Andersson AF. 2011. Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. ISME J. 5: 1571-1579.
DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, et al. 2006. Greengenes, a chimerachecked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 72: 5069-5072.
Benson AK, Kelly SA, Legge R, Ma FR, Low SJ, Kim J, et al. 2010. Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors. P. Natl. Acad. Sci. USA 107: 18933-18938.
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